PLOS ONE

Late ninth millennium B.P. use of Zea mays L. at Cubiln, highland Ecuador, revealed
by ancient starch grains
--Manuscript Draft-Manuscript Number:

PONE-D-14-48208

Article Type:

Research Article

Full Title:

Late ninth millennium B.P. use of Zea mays L. at Cubiln, highland Ecuador, revealed
by ancient starch grains

Short Title:

Earliest evidence for Zea mays L. use in South America

Corresponding Author:

Jaime R. Pagn-Jimnez, Ph.D.

Instituto Nacional de Patrimonio Cultural
Quito, Pichincha ECUADOR

Keywords:

maize; domestication; plant dispersals; Andes; highlands; South America; starch

grains; lithic tools; Preceramic; Cubiln; paleoethnobotany; archaeobotany; ancient
phytogeography; manioc; wild yam; chili pepper; Marantaceae; Sagittaria spp.;
Neotropics; Azuay; Loja; Ecuador

Abstract:

Today, maize is acknowledged as a plant with a great culinary and industrial versatility.
It also has a deep relationship with the native cultures of the Americas and is still a vital
food source for hundreds millions of people worldwide. By means of starch grain
extraction from ancient lithic tools used more than 8000 years ago, here we report what
is so far the oldest documented occurrence of maize in South America. This study
places maize in the Ecuadorian highlands during a period coinciding with the initial
stage of maize diversification and long distance expansion after its domestication in
southwestern Mexico. These results allow us to unravel an early episode of human
innovation previously unknown for South America. This and other similar episodes of
early phytocultural innovation in the region are the first steps toward the full re-shaping
of human subsistence strategies at the whole continent.

Order of Authors:

Jaime R. Pagn-Jimnez, Ph.D.

Martha E. Romero-Bastidas
Ana M. Guachamn-Tello
Angelo R. Constantine-Castro

Suggested Reviewers:

Mara del Pilar Babot, Ph.D.

Researcher, Universidad Nacional de Tucuman
pilarbabot@yahoo.com
Mara L. Pochettino, Ph.D.
Researcher, Universidad Nacional de la Plata
pochett@fcnym.unlp.edu.ar
Jos Iriarte, Ph.D.
Professor, University of Exeter
J.Iriarte@exeter.ac.uk
Linda Perry, Ph.D.
Researcher, The Farm, USA
linda.at.the.farm@gmail.com

Opposed Reviewers:
Additional Information:
Question

Response

Financial Disclosure

This research was supported by a grant from Proyecto Prometeo, Secretara Nacional
de Educacin Superior, Ciencia, Tecnologa e Innovacin (SENESCYT, Ecuador) to
Pagn-Jimnez, and by another grant from SENESCYT to the Instituto Nacional de
Patrimonio Cultural, Ecuador. The funders had no role in study design, data collection

Powered by Editorial Manager and ProduXion Manager from Aries Systems Corporation

Please describe all sources of funding

and analysis, decision to publish, or preparation of the manuscript.
that have supported your work. A
complete funding statement should do the
following:
Include grant numbers and the URLs of
any funder's website. Use the full name,
not acronyms, of funding institutions, and
use initials to identify authors who
received the funding.
Describe the role of any sponsors or
funders in the study design, data
collection and analysis, decision to
publish, or preparation of the manuscript.
If they had no role in any of the above,
include this sentence at the end of your
statement: "The funders had no role in
study design, data collection and analysis,
decision to publish, or preparation of the
manuscript."
If the study was unfunded, provide a
statement that clearly indicates this, for
example: "The author(s) received no
specific funding for this work."

* typeset
Competing Interests

The authors have declared that no competing interests exist.

You are responsible for recognizing and

disclosing on behalf of all authors any
competing interest that could be
perceived to bias their work,
acknowledging all financial support and
any other relevant financial or nonfinancial competing interests.

Do any authors of this manuscript have

competing interests (as described in the
PLOS Policy on Declaration and
Evaluation of Competing Interests)?

If yes, please provide details about any

and all competing interests in the box
below. Your response should begin with
this statement: I have read the journal's
policy and the authors of this manuscript
have the following competing interests:

If no authors have any competing

interests to declare, please enter this
statement in the box: "The authors have
declared that no competing interests
exist."
Powered by Editorial Manager and ProduXion Manager from Aries Systems Corporation

* typeset
Ethics Statement

You must provide an ethics statement if

your study involved human participants,
specimens or tissue samples, or
vertebrate animals, embryos or tissues.
All information entered here should also
be included in the Methods section of your
manuscript. Please write "N/A" if your
study does not require an ethics
statement.

Ecuadors Decree No. 2600 of 9 June 1978 created the Instituto Nacional de Patrimoio
Cultural (INPC). The INPC regulates the performance of anthropological studies,
including archaeology, in Ecuador. As such, the INPC is the lead Ecuadorian authority
regulating the practice of archaeology and is charged by law for safeguarding the
archaeological heritage of the nation. No permits were required for its own Laboratorio
de Investigacin for conducting the field and laboratory studies here described.

Human Subject Research (involved

human participants and/or tissue)
All research involving human participants
must have been approved by the authors'
Institutional Review Board (IRB) or an
equivalent committee, and all clinical
investigation must have been conducted
according to the principles expressed in
the Declaration of Helsinki. Informed
consent, written or oral, should also have
been obtained from the participants. If no
consent was given, the reason must be
explained (e.g. the data were analyzed
anonymously) and reported. The form of
consent (written/oral), or reason for lack of
consent, should be indicated in the
Methods section of your manuscript.

Please enter the name of the IRB or

Ethics Committee that approved this study
in the space below. Include the approval
number and/or a statement indicating
approval of this research.

Animal Research (involved vertebrate

animals, embryos or tissues)
All animal work must have been
conducted according to relevant national
and international guidelines. If your study
involved non-human primates, you must
provide details regarding animal welfare
and steps taken to ameliorate suffering;
this is in accordance with the
recommendations of the Weatherall
report, "The use of non-human primates in
research." The relevant guidelines
followed and the committee that approved
the study should be identified in the ethics
statement.
Powered by Editorial Manager and ProduXion Manager from Aries Systems Corporation

If anesthesia, euthanasia or any kind of

animal sacrifice is part of the study,
please include briefly in your statement
which substances and/or methods were
applied.

Please enter the name of your Institutional

Animal Care and Use Committee (IACUC)
or other relevant ethics board, and
indicate whether they approved this
research or granted a formal waiver of
ethical approval. Also include an approval
number if one was obtained.

Field Permit
Please indicate the name of the institution
or the relevant body that granted
permission.
Data Availability

Yes - all data are fully available without restriction

PLOS journals require authors to make all

data underlying the findings described in
their manuscript fully available, without
restriction and from the time of
publication, with only rare exceptions to
address legal and ethical concerns (see
the PLOS Data Policy and FAQ for further
details). When submitting a manuscript,
authors must provide a Data Availability
Statement that describes where the data
underlying their manuscript can be found.
Your answers to the following constitute
your statement about data availability and
will be included with the article in the
event of publication. Please note that
simply stating data available on request
from the author is not acceptable. If,
however, your data are only available
upon request from the author(s), you must
answer No to the first question below,
and explain your exceptional situation in
the text box provided.
Do the authors confirm that all data
underlying the findings described in their
manuscript are fully available without
restriction?
Please describe where your data may be All relevant data are within the paper and its Supporting Information files.
found, writing in full sentences. Your
answers should be entered into the box
below and will be published in the form
you provide them, if your manuscript is
accepted. If you are copying our sample
text below, please ensure you replace any
Powered by Editorial Manager and ProduXion Manager from Aries Systems Corporation

instances of XXX with the appropriate

details.

If your data are all contained within the

paper and/or Supporting Information files,
please state this in your answer below.
For example, All relevant data are within
the paper and its Supporting Information
files.
If your data are held or will be held in a
public repository, include URLs,
accession numbers or DOIs. For example,
All XXX files are available from the XXX
database (accession number(s) XXX,
XXX)." If this information will only be
available after acceptance, please
indicate this by ticking the box below.
If neither of these applies but you are able
to provide details of access elsewhere,
with or without limitations, please do so in
the box below. For example:
Data are available from the XXX
Institutional Data Access / Ethics
Committee for researchers who meet the
criteria for access to confidential data.
Data are from the XXX study whose
authors may be contacted at XXX.
* typeset
Additional data availability information:

Powered by Editorial Manager and ProduXion Manager from Aries Systems Corporation

Cover Letter

Quito, Ecuador, 27 October 2014

Dr Damian Pattinson
Editorial Director, PLOS ONE
San Francisco, CA, USA
Dear Dr Pattinson,
We have unexpectedly discovered microbotanical evidence representing the oldest
documentation of maize use in South America. Our starch grain analysis places maize in the
Ecuadorian highlands during the initial stage of maize diversification and long distance
expansion after its domestication in southwestern Mexico. This finding could change prime
notions of current archaeology, paleoethnobotany, and genetic research that deal with the
elucidation of early maize domestication and its dispersal throughout the Western hemisphere.
Our results exemplify how a multiproxi starch grain approach is fruitful for building new
perspectives on early peoples of the Americas and their relation to maize's evolutionary history.
Today, maize is acknowledged for being a plant with a great culinary and industrial versatility.
It has a deep relationship with the native cultures of the Americas and is still a vital food source
for millions of people worldwide. Armed with fresh archaeobotanical (starch grain) evidence,
we communicate our findings of maize by unraveling an early episode of human innovation
previously unknown for the Western hemisphere that goes back to 8,078 cal. years before
present. The setting for this enthralling adventure between humans and maize are the
Ecuadorian Andes, a region where people are aware of their role in the ethnobotanical history of
humankind.
Our prospective contribution (research article) to your journal is entitled Late ninth millennium
B.P. use of Zea mays L. at Cubiln, highland Ecuador, revealed by ancient starch grains. We
proposed also a short title for our contribution: Earliest evidence for Zea mays L. use in South
America.
We suggest Dr. John P. Hart or Dr. Karen Hardy as Academic editors for handling our
manuscript or for formally revising our paper. They has deep knowledge on maize
domestication/early dispersals and on starch grain analysis applied to archaeological research.
Potential referees for this manuscript will be Dr. Linda Perry, The Farm (USA)
linda.at.the.farm@gmail.com; Dr. Maria del Pilar Babot, Instituto de Arqueologa, Universidad
Nacional de Tucumn (Argentina), pilarbabot@yahoo.com; Dr. Jos Iriarte, Department of
Archaeology, University of Exeter (United Kingdom) J.Iriarte@exeter.ac.uk; and Dr. Maria L.
Pochettino, Laboratorio de Etnobotnica y Botnica Aplicada, Universidad Nacional de La Plata
(Argentina) pochett@fcnym.unlp.edu.ar.
We hope this work will meet the expectations and requirements of the important scientific
leading journal you head.
Sincerely,
Dr Jaime R. Pagn-Jimnez (Corresponding author)
Instituto Nacional de Patrimonio Cultural
Av. Coln Oe1-93 y Av. 10 de Agosto (La Circasiana)
Quito, Ecuador, EC170129
Work phone: (593-2) 222 7927, Extension 117
Email: jpaganpr@yahoo.com

Manuscript
Click here to download Manuscript: JRPJ-manuscript.docx

Full Title:
Late ninth millennium B.P. use of Zea mays L. at Cubiln, highland
Ecuador, revealed by ancient starch grains
Short Title:

Earliest evidence for Zea mays L. use in South America

Jaime R. Pagn-Jimnez1*, Martha E. Romero-Bastidas1, Ana M. Guachamn-Tello1,

Angelo R. Constantine-Castro2

Laboratorio de Investigacin, Instituto Nacional de Patrimonio Cultural. Ave. Coln Oe1-93

y Ave 10 de Agosto, Quito, Ecuador.

2

Grupo de Estudios Geoarqueolgicos de Amrica Latina. Urb. Puerto Azul, MZ A2,

Condominio Paola Vernica, Guayaquil, Ecuador.

*Corresponding author
Email: jpaganpr@yahoo.com (JPJ)

These authors contribute equally to this work.

Abstract
Today, maize is acknowledged as a plant with a great culinary and industrial
versatility. It also has a deep relationship with the native cultures of the Americas and is still a
vital food source for hundreds millions of people worldwide. By means of starch grain
extraction from ancient lithic tools used more than 8000 years ago, here we report what is so
far the oldest documented occurrence of maize in South America. This study places maize in
the Ecuadorian highlands during a period coinciding with the initial stage of maize
diversification and long distance expansion after its domestication in southwestern Mexico.
These results allow us to unravel an early episode of human innovation previously unknown
for South America. This and other similar episodes of early phytocultural innovation in the
region are the first steps toward the full re-shaping of human subsistence strategies at the
whole continent.

Introduction
During the last two decades, archaeobotanical and genetic research on the origin and
dispersal of maize (Zea mays ssp. mays) has generated significant data on its initial spread as
a full domesticate from its lowland source area in southwestern Mexico between 10000 to
8600 cal. years ago [1,2]. However, its earliest dispersals southward have only been partially
reconstructed by means of microbotanical data recovered from lowland archaeological sites
and paleoenvironmental analyses [39]. Here we present the results of recent microbotanical
studies from Cubiln site #2 (Cu-S2) in highland Ecuador (Figure 1), where we recovered
maize starches from milling and scrapping lithic tools associated with contexts of 8078 - 7959
cal. yrs. B.P. Thus far, this is the oldest evidence of maize in South America. Starch grains
from other low to mid-elevation plants such as manioc (Manihot esculenta), chili pepper

(Capsicum sp.), wild yam (Dioscoreaceae) and wild Calathea sp. were also identified. This
evidence clearly shows that the early inhabitants of Cubiln were engaged in the cultivation of
maize and other useful plants in the highlands or in the neighboring midlands.
As part of an ongoing nationwide research program on the paleoethnobotany of
Ecuadors ancestral cultures carried out by the Instituto Nacional de Patrimonio Cultural
(INPC), archaeological prospection and test excavations were conducted at four previously
unknown highland archaeological localities known as the Cubiln sites [10] in Oa, Provincia
de Azuay, southern Ecuador. The Cubiln sites cover an area of ~30 km2 where previous
studies identified 24 other archaeological localities, characterizing two of them as lithic
workshops or camp sites of hunter-gatherer societies that occupied the region by ~12700 to
9900 cal yrs. B.P. [10]. Site Cu-S2 is located within the Jubones river watershed, an irregular
mountainous area located between 2400 to 3150 m above sea level (asl). The topography of
that area consists of narrow and sometimes partially flat ridges, and small elongated valleys
surrounded by hills and old fluvial terraces. At present, Cu-S2 is in the very humid montane
forest that falls between two contrasting ecological zones [11]: the very dry tropical forest ~5
km to the northwest, and the very humid pre-montane forest ~13 km to the southeast.
Pramo herbs such as Calamagrostis spp. dominate the open vegetation of Cu-S2 and
its surroundings, though pramo shrubs are also common. Discrete patches of primary humid
montane forest are located on the east and west side of steep slopes which serves to protect
them from constant and sometimes strong winds that come from the Amazon Basin.
However, by around 8000 cal yrs B.P. palaecological records for southern Ecuadorian pramo
have shown a marked increase in charcoal influx that has been related to human-caused fires
intentionally set to clear lands, to dryer and warmer climatic conditions, and to an expansion
of the forest to higher altitudes when compared to current conditions registered at Cubiln
area [12,13]. This would explain the modern patchy distributional pattern of humid montane

forests remnants within protective slopes at nearby altitudes. Cultivation practices or human
settlements have not been recorded in recent times for the study area and there are no
ethnographic and historical data which could place any kind of plant production activities,
present or in the recorded past, at Cu-S2 or its immediate periphery. However, some small
river banks and fluvial terraces found in a radius of 2 to 4 Km west of Cu-S2, at elevations
between 2600 to 3000 masl located at the bottom of those protective slopes, has potentially
good soils and warmer conditions suitable for the cultivation of several high altitude edible
plants such as potato (Solanum tuberosum), melloco (Ullucus tuberosus), maize, and some
legumes. Other production practices such as sporadic cattle grazing, have been confirmed for
this pramo area, though owners of these communal lands abandoned this practice several
years ago.

Sample provenance and methods

Surface collection data from the study area revealed lithic debitage, and formalized
and expedient flaked tools (e.g., scrapers, projectile points, bifaces) produced by bipolar and
free-hand flaking. These data are consistent with typical Ecuadorian preceramic technologies
of lithic production [14,15]. No pre-Columbian ceramic age or modern artifacts were found at
Cu-S2. Two 2 x 2m test units at this locality produced 10,922 preceramic artifacts, mostly
siliceous lithic debitage (n=10,192). Ten lithic tools selected for this analysis (Figure S1) are
seven formalized scrapers, two unmodified flakes with marginal use-wear consistent with
scraping activities, and one lightly utilized rudimentary milling stone base fragment (Table
1).These ten tools were from stratum III and underlying stratum II (strata numbered I-IV,
bottom to top, Figure 2). Stratum IV did not yield archaeological artifacts. The middle and
lower sections of stratum III in Units 1 and 2, both units separated by 2 m, where a single
occupational surface was detected, were sampled for 14C dating resulting in two statistically
4

overlapped dates between 80787959 cal yrs. B.P. (Table 2). Four of the studied tools came
from this stratum III, while the other six were from the underlying stratum II, thus confirming
that any tool or starch grains from stratum III or lower falls within, or earlier than these
calibrated dates. Two more dates (9463 9233 cal. yrs. B.P., and 1630915878 cal. yrs. B.P.)
have been temporarily excluded from this analysis because they clearly predate the estimate
for the onset of maize domestication in upper Central America [1,2]. Five tools that yield
maize starches from upper stratum II (14-02, 14-05), and from middle and lower stratum II
(14-04, 13-98 and 13-99), were all located near the provenance of these excluded dates.
Interestingly, older occupational deposits [10] corresponding to the same stratigraphic units
here defined for Cu-S2 (stratum IV and III), and buried at about 20 to 35 cm below surface,
were detected and dated at sites Cu-26 and Cu-27 (Table 2), thus suggesting a very low
accumulation rate of organic soils in these early archaeological localities of the southern
Ecuadorian pramo. This is consistent with other palaeoecological data chronologically
placed from the early to mid-Holocene (~ 8000 cal yrs. B.P.) in the region [13].

Artifact management, sample extraction and starch grain

recovery
Artifacts selected for this study were isolated from the field and placed individually
and unwashed in new plastic bags. Standard criteria were used for lithics descriptions [16].
Methods for sediment extraction from lithic tools [2], and starch separation from sediments
[17] were modified from others published elsewhere, as shown below. Once in the lab,
artifacts were carefully rinsed to gently remove any modern potential contaminant and then
were placed separately in new and sterile zip lock bags, adding enough water to cover each
artifact. Each bagged artifact was then placed into an ultrasonic bath for 40 minutes in order
to release sediments from their used margins or surfaces, pores and cracks. Released sediment
5

or residues were placed in new and sterile 50 ml. centrifuge vials, and then centrifuged at
3500 rpm x 15 minutes. Water was decanted and sample vials were placed in a lab oven at
31 C for 60 hrs. until sediments were completely dried. A CsCl solution with a specific
gravity of 1.79 was added to each sample, sufficient enough to cover over the solid sediments
in the vials by 4cm. Vials were agitated manually for 20 seconds, and then more CsCl was
used release sediments and residues from the walls and lids of the vials. Centrifugation
proceeded later at 3000 rpm for 20 minutes and all the solution with the supernatant was
transferred into new and sterile centrifuge vials. A second round of CsCl flotation was
executed with the original samples in their own vials in order to catch more residues that
could remain trapped within the matrix. The resulting solution and supernatant was also
placed in the previously used centrifuge vials where the solution of the first round flotation
was stored. Sufficient bi-distilled water was added to each sample to break down the 1.79
density of the CsCl solution. In this way, starches and other residues would be forced to sink
to the bottom of the vials. Centrifugation at 4000 rpm was executed for 20 minutes and then
the undesirable supernatant was decanted. The previous step of adding more water to each
vial was executed four more times for diluting the remaining salts of the solution while
retaining the residues at the bottom of the vials. Once the solution with the residue samples
was completely washed from inside each 50 ml. vial they were transferred into various new
and sterile 2.0 ml. microcentrifuge tubes for each sample. Bi-distilled water was added to each
microcentrifuge tube and then centrifuged at 8000 rpm for 10 minutes. After that, the water at
the top of each tube was taken off and the remaining samples (at the bottom of the tubes) were
allowed to dry in isolation at 30C inside a lab oven. Then, sample mounting into microscope
slides proceeded (see below).
Throughout the washing steps described above, the discarded solution was repeatedly
tested to check to be sure that we were not losing ancient starch grains; no starch grains were

detected at any point during the process. Following Zarrillo [18] recommendations, a major
concern of this study was to test if modern starches were potential contaminant agents of the
archaeological samples. To assess this, we submitted all the materials used during the
extraction and flotation process to close examination for starch grains. Unpowdered nitrile
gloves, glass beakers, new disposable pipettes, new zip lock bags, new and sterile centrifuge
vials, new microscope slides and cover slips, as well as solutions and reagents such as glycerol,
unused CsCl, and bi-distilled water were all sampled separately looking for potential modern
starch grain contamination, following standard microscope sample mounting procedures. This
testing resulted negative to the presence of contaminant starches. As a further precaution, new
microscope slides and cover slips were washed with bi-distilled water and allowed to dry in
isolation at 80C inside a lab oven [18]. These materials were then placed in a plastic
microscope slide holder that was previously washed and tested for possible modern starch
contamination.
Lithic tools analyzed for this study, and samples extracted from them, have been
identified with a unique code as shown in Table 1 (see Sample no. column). All of the tools
recovered and the pertinent documentation for the CU-S2 studies are permanently curated at
the INPC in Quito. Please note that Ecuadors Decree No. 2600 of 9 June 1978 created the
INPC. The INPC regulates the performance of anthropological studies, including archaeology,
in Ecuador. As such, the INPC is the lead Ecuadorian authority regulating the practice of
archaeology and is charged by law for safeguarding the archaeological heritage of the nation.
No permits were required for its own Laboratorio de Investigacin for conducting the field and
laboratory studies. However, the field and laboratory studies performed for this research
strictly adhered to INPCs national regulatory framework such as the Reglamento para la
Concesin de Permisos de Investigacin Arqueolgica Terrestre of 20 February 1992, in

compliance with national and international academic, professional and ethical standards of the
discipline.

Comparative criteria for starch grain identification

Starch identification used 140 modern neotropical and Andean starchy plant
specimens from the Laboratorio de Investigacin, INPC (LIINPC), Pagn-Jimnez reference
collections, and other published works [7,17,19,20]. Taxonomical ascription of archaeological
starch grains consisted of their comparison to a reference collection stored at the LIINPC
made up by 63 neotropical specimens that comprise 23 plant families, 26 genera, and 37
species which includes 13 different native maize landraces and 7 different Phaseolus spp.
varieties, among other important economic and wild plants of Ecuador. Additionally, we used
Pagn-Jimnez reference collection of pan-tropical starchy plants that include 77 specimens
comprising 68 genera and 61 species, in which 6 different Circum-Caribbean native maize
landraces are represented. Published literature on diagnostic criteria for starch grains was also
consulted [2,5,7,9,1929], whereas damaging patterns observed in the recovered starches
were contrasted to our own published work or to previously published literature [2933].
Secure and tentative identifications of starches in this study are based on diagnostic and/or
distinctive features described elsewhere. Specific morphometric features used are shape, size,
presence and location of the hilum within the granule, presence and appearance of fissures,
presence and type of pressure facets, presence and appearance of lamellae, and in some cases
the appearance and projection of the Maltese cross.
Microscope bi-dimensional analysis consisted first in placing sample drops on
microscope slides (sometimes up to 4 preparations per sample), and then mixing it with
glycerol for adding viscosity to the media, which allow controlled movement and rotation of
the found starches. Cover slips were placed and later microscope examination began. We used

an Olympus BX53optical microscope with polarized capacity and two different digital
cameras: an Olympus DP26-CU, and an Infinity 1-2CB with their respective software. Every
slide was surveyed entirely with a 10X objective, starches found were photographed, and 12
morphometric variables were recorded for each of them with a 40X objective. Finally, their
position was registered with coordinates for allowing further revisions, and microscope slides
were stored in new cardboard slide holders.

Results and discussion

The identification at Cu-S2 (Figure 3), of 103 individual, plus ~68 clustered starch
grains, all from maize were fully or tentatively identified using diagnostic criteria previously
published [17,19,21,28,29] and confirmed by our own modern maize reference collection at
the LIINPC (Table 3). Starches from other important potential food plants such as beans
(Fabaceae, Phaseolus spp.), yams (Dioscoreaceae), calathea (Calathea spp.), manioc, Indian
arrowhead (Sagittaria spp.), and chili pepper (Capsicum spp.), either from mid-elevations or
the lowland, were also identified in the same dated contexts (Table 4), adding potential
information on poorly understood interregional phytocultural interactions between the South
American lowlands and the highlands (Figures S2 to S7). Table 5 shows specific bidimensional and morphometric data on tentative and secure maize starch grain identifications.
Maize starches were recovered on all ten tools, but more noticeable in those two recovered
between 50 to 70cmbd in Unit 1. Tool 13-98, a rudimentary grinding tool fragment, produced
maize starches that show damaging features highly consistent with pressure
(grinding/pounding), such as pronounced radial fissures/striations, as well as light to heavy
modifications on its common shapes, documented in the heaviest tools 13-98 and 13-99 [29].
Another clear pressure-damage indicator, noted previously in experimental maize
starches, is the direct relationship that exists between kernel hardness and starch enlargement
9

due to differential kernel resistance during intensive grinding [29]. Neither maize starch of the
tools discussed above, nor those recovered on the other 8 scrapers, show size ranges that fit
any experimentally documented starches produced during grinding (Figure 4). However, they
match nicely with four modern maize landraces with flint or floury endosperm from Andean
mid- to high elevations (cf. Mishca, Blanco Blandito redondeado, Morochillo, Tusilla) that
were not experimentally modified. Tusilla, a mid-elevation maize with flint/hard endosperm is
the only one that produces mostly irregular starches clearly comparable to those documented
on all ten tools. Because maize endosperm formation and amylose synthesis are both
genetically controlled [34], the morphometric features of maize starches identified on the
tools suggest that the maize processed and used in Cubiln was one with hard endosperm and
genotypic characteristics comparable to modern Tusilla landrace. Interestingly, hard
endosperm maize was previously suggested as the earliest domesticated type of maize for
Mexico [2] on the basis of recovered ancient starch grains similar to the ones identified here.
Eight of the analyzed tools are relatively small formalized or expedient scrapers.
Although they contained starches with signs of damage caused by pressure, the damage
occurs at a lower percent than in the larger grinding and scraping tools. Previous studies
clearly indicate that when totally green or immature flint maize kernels are processed with
these types of tools, the starches do not undergo great morphometric transformation.
Conversely, when mature partially dry maize kernels, or totally dry unsoaked kernels were
processed experimentally, then the starch undergoes significantly greater morphometric
transformations during grinding than that observed in our archaeologically recovered
starches[29]. On this basis, we suggest that the maize intentionally processed with the
grinding and scraping tools at Cu-S2 was likely in a mature state, still conserving a soft
(possibly pasty) interior matrix.

10

In summary, we suggest that in Cu-S2 the grinding tool was used for intentionally
grinding mature, though flinty maize kernels, whereas scrapers were used for peeling and
releasing mature kernels that were heavily attached to maize cobs. These processing methods
could be related to transforming maize into wrapped masses (jumint'a or tamal), or for
preparing energetic or alcoholic beverages derived from doughy masses. It is evident that
kernels were the main focus of processing and use of maize in Cubiln, contrary to
hypotheses suggesting sugary stalks were the main reason for maize domestication and its
early dispersal [35]. Other cultural factors surrounding the preparation and consumption of
food or beverages could govern the forms of intentional use of the maize documented here.
The processing and manipulation of maize derivatives in what has been preliminary
interpreted as a lithic workshop site indicates that this location constituted a multipurpose
setting for performing varied, but culturally interrelated social activities.
Although some evidence suggests that the earliest domestic maize was originally
adapted to the humid Mexican lowlands [2,36], our findings fit various genetic-derived
hypotheses which indicate that the highlands constituted fundamental settings for early maize
domestication and pathways for its dispersal [1,37]. Maize germplasm in the form of seeds
were present at the highland locality of Cu-S2 and it seems that they were processed when
they were already mature or partially fresh. Having maize seeds at hand, it is not surprising
that the inhabitants of Cu-S2 could produce maize plants experimentally [38,39] on-site when
climate was warmer and dryer by ~ 8000 cal. yrs. B.P. according to palaeoecological data, or
in nearby lower physiographic strata within a radius of ca. 5 to 13 km. Interestingly
preceding phytolith data has pointed out that maize reached the seasonally dry forests of the
coastal lowlands of Ecuador

as egas-

E-80 at

8053 7818 cal. yrs. B.P.[4], while

recently gathered starch grain data has shown that humans also dispersed maize into the midelevation (1650 masl) and very humid premontane forests of the Colombian Andes in

11

contexts dating to 80007600 cal. yrs B.P.[40]. This seemingly contrasting information,
together with new data presented here for Cu-S2, suggests that distinct maize landraces
formerly adapted to different altitudinal strata and climatic conditions could have entered and
dispersed simultaneously from northwestern South America towards other continental regions
in a statistically overlapping time period between 81007900 cal. yrs. B.P.
The identification of starches from other plants adapted to mid- and low elevations on
some of the studied tools allows us to infer that the inhabitants of Cu-S2 operated within a
geographical wide-spectrum range of exploitation. It is foreseeable that the inhabitants of
Cubiln made use of some of the nearby diverse and contrasting physiographic environments
to grow plants like maize, or to grow and extract other vegetal resources from these zones.
Though, we cannot completely rule out the possibility that the identified plants may have
been acquired through exchange networks that extended across different altitudinal
environments. The significant density of ancient archaeological localities in the Ecuadorian
lowlands and highlands has recently been recognized [14,15] raising the possibility that the
early peopling of the Ecuadorian territory followed active and preferred circuits for mobility
and intensive human interaction that included the exchange of goods, products and ideas. This
socio-spatial ingredient, possibly developed since the Pleistocene-Holocene transition in
northwestern South America [41], could be the driving force that later triggered early long
distance dispersion of cultivars and domestic plants betwixt and between Central and South
America, which had, as one of its main corridors, the Ecuadorian highlands.

Acknowledgments
Thank you is due to Jeff Walker, Reniel Rodrguez-Ramos, Carlos Sols-Magaa, and
James D. Ackerman for editing our English translation of the article.

12

Author contributions
Conceived and designed the research, processed lithic tools and extracted samples for starch
analysis: JRPJ. Collected modern Ecuadorian specimens of maize and performed the
respective starch grain morphometrical analysis: JRPJ, AMGT and MERB. Provided reagents
and equipment for conducting lab research: MERB and AMGT. Performed field work,
collected lithic artifacts, and analyzed preliminary archaeological results: ARCC. Wrote the
paper: JPJ.

References
1.

Matsuoka Y, Vigouroux Y, Goodman MM, Sanchez G J, Buckler E, et al. (2002) A

single domestication for maize shown by multilocus microsatellite genotyping.
Proceedings of the National Academy of Science of the United States of America 99:
60806084. doi:10.1073/pnas.052125199.

2.

Piperno DR, Ranere AJ, Holst I, Iriarte J, Dickau R (2009) Starch grain and phytolith
evidence for early ninth millennium B.P. maize from the Central Balsas River Valley,
Mexico. Proceedings of the National Academy of Science of the United States of
America 106: 50195024. doi:10.1073/pnas.0812525106.

3.

Grobman A, Bonavia D, Dillehay TD, Piperno DR, Holst I (2011) Preceramic maize
from Paredones and Huaca Prieta, Per. Proceedings of the National Academy of
Science of the United States of America 109: 17551759.
doi:10.1073/pnas.1120270109.

4.

Piperno DR (2011) The Origins of Plant Cultivation and Domestication in the New
World Tropics. Current Anthropology 52: S453S470. doi:10.1086/659998.
13

5.

Zarrillo S, Pearsall DM, Raymond JS, Tisdale MA, Quon DJ (2008) Directly dated
starch residues document early formative maize (Zea mays L .) in tropical Ecuador.
Proceedings of the National Academy of Science of the United States of America 105:
50065011.

6.

Pearsall DM (2008) Plant Domestication and the Shift to Agriculture in the Andes. In:
Silverman H, Isbell W, editors. The Handbook of South American Archaeology. New
York: Springer. pp. 105120.

7.

Piperno DR, Ranere A, Holst I, Hansell P (2000) Starch grains reveal early root crop
horticulture in the Panamanian tropical forest. Nature 407: 894897.

8.

Stothert KE, Piperno DR, Andres CT (2003) Terminal Pleistocene/Early Holocene

Human Adaptation in Coastal Ecuador: The Las Vegas Evidence. Quaternary
International 109-110: 2343.

9.

Dickau R, Ranere AJ, Cooke RG (2007) Starch grain evidence for the preceramic
dispersals of maize and root crops into tropical dry and humid forests of Panama.
Proceedings of the National Academy of Science of the United States of America 104:
36513656.

10.

Temme M (2005) Cubiln: dos estaciones precermicas en el curso superior del rio San
Felipe de Oa, Provincias de Loja y Azuay, Ecuador. Loja: Universidad Tcnica
Particular de Loja.

11.

Ecuador Repblica del (2011) Mapa Ecolgico. Quito: Ministerio de Agricultura,

Ganaderia, Acuacultura y Pesca.

14

12.

Villota A (2012) Vegetation, climate, and fire history of the Pramo of Jimbura,
southern Ecuador. Quaternary International 279-280: 521.
doi:10.1016/j.quaint.2012.08.1809.

13.

Jantz N, Behling H (2011) A Holocene environmental record reflecting vegetation,

climate, and fire variability at the Pramo of Quimsacocha, southwestern Ecuadorian
Andes. Vegetational History and Archaeobotany 21: 169185. doi:10.1007/s00334011-0327-x.

14.

Stothert KE, Snchez A (2011) Culturas del Pleistoceno final y el Holoceno temprano
en el Ecuador. Boletn de Arqueologa de la Pontificia Universidad Catlica del Per:
81119.

15.

Constantine A (2013) The early settlement of Continental Ecuador: New evidence from
preceramic sites in the Tropical Rain Forest. Quaternary International 317: 112117.

16.

Ranere AJ, Piperno DR, Holst I, Dickau R, Iriarte J (2009) The cultural and
chronological context of early Holocene maize and squash domestication in the Central
Balsas River Valley, Mexico. Proceedings of the National Academy of Science of the
United States of America 106: 50145018. doi:10.1073/pnas.0812590106.

17.

Pagn-Jimnez JR (2007) De antiguos pueblos y culturas botnicas en el Puerto Rico

indgena. El archipilago borincano y la llegada de los primeros pobladores
agroceramistas. Oxford: Paris Monographs in American Archaeology 18, BAR
International Series, Archaeopress.

15

18.

Zarrillo S (2012) Human Adaptation, Food Production, And Cultural Interaction during
the Formative Period in Highland Ecuador. Calgary: PhD Dissertation. Department of
Archaeology, University of Calgary.

19.

Holst I, Moreno JE, Piperno DR (2007) Identification of teosinte , maize , and

Tripsacum in Mesoamerica by using pollen , starch grains , and phytoliths. Proceedings
of the National Academy of Science of the United States of America 104: 17608
17613.

20.

Perry L, Sandweiss DH, Piperno DR, Rademaker K, Malpass MA, et al. (2006) Early
maize agriculture and interzonal interaction in southern Per. Nature 440: 7679.
doi:10.1038/nature04294.

21.

Pearsall DM, Chandler-Ezell K, Zeidler JA (2004) Maize in ancient Ecuador : results

of residue analysis of stone tools from the Real Alto site. Journal of Archaeological
Science 31: 423442.

22.

Piperno DR, Dillehay TD (2008) Starch grains on human teeth reveal early broad crop
diet in northern Peru. Proceedings of the National Academy of Science of the United
States of America 105: 1962219627.

23.

Piperno DR, Holst I (1998) The Presence of Starch Grains on Prehistoric Stone Tools
from the Humid Neotropics : Indications of Early Tuber Use and Agriculture in
Panama. Journal of Archaeological Science 25: 765776.

24.

Perry L, Dickau R, Zarrillo S, Holst I, Pearsall DM, et al. (2007) Starch fossils and the
domestication and dispersal of chili peppers (Capsicum spp. L.) in the Americas.
Science 315: 986988. doi:10.1126/science.1136914.

16

25.

Perry L (2004) Starch granule size and the domestication of manioc (Manihot
esculenta) and sweet potato (Ipomoea batatas). Economic Botany 56: 335349.

26.

Reichert ET (1913) The Differentiation and Specificity of Starches in Relation to

Genera, Species, etc. Washington D.C.: Carnegie Institution of Washington.

27.

Horrocks M, Rechtman RB (2009) Sweet potato ( Ipomoea batatas ) and banana (Musa
sp .) microfossils in deposits from the Kona Field System , Island of Hawaii. Journal of
Archaeological Science 36: 11151126. doi:10.1016/j.jas.2008.12.014.

28.

Musaubach MG, Plos A, Babot M del P (2013) Differentiation of archaeological maize

(Zea mays L .) from native wild grasses based on starch grain morphology. Cases from
the Central Pampas of Argentina. Journal of Archaeological Science 40: 11861193.

29.

Mickleburgh HL, Pagn-Jimnez JR (2012) New insights into the consumption of

maize and other food plants in the pre-Columbian Caribbean from starch grains trapped
in human dental calculus. Journal of Archaeological Science 39: 24682478.

30.

Babot M del P (2003) Starch grain damage as an indicator of food processing. In: Hart
DM, Wallis LA, editors. Phytolith and starch research in the Australian- Pacific-Asian
regions: the state of the art. Canberra: Pandanus Books. pp. 6981.

31.

Henry AG, Hudson HF, Piperno DR (2009) Changes in starch grain morphologies from
cooking. Journal of Archaeological Science 36: 915922.

32.

Lamb J, Loy T (2005) Seeing red : the use of Congo Red dye to identify cooked and
damaged starch grains in archaeological residues. Journal of Archaeological Science
32: 14331440. doi:10.1016/j.jas.2005.03.020.

17

33.

Piperno DR, Weiss E, Holst I, Nadel D (2004) Processing of wild cereal grains in the
Upper Palaeolithic revealed by starch grain analysis. Nature 430: 670673.

34.

Smith AM, Denyer K, Martin CR (1995) What Controls the Amount and Structure of
Starch in Storage Organs? Plant Physiology 107: 673677.

35.

Smalley J, Blake M (2003) Sweet beginnings: Stalk sugar and the domestication of
maize. Current Anthropology 44: 675703.

36.

Van Heerwaarden J, Doebley J, Briggs WH, Glaubitz JC, Goodman MM, et al. (2011)
Genetic signals of origin, spread, and introgression in a large sample of maize
landraces. Proceedings of the National Academy of Science of the United States of
America 108: 10881092. doi:10.1073/pnas.1013011108.

37.

Lia V V., Confalonieri V, Ratto N, Cmara JA, Miante Alzogaray AM, et al. (2006)
Microsatellite typing of ancient maize: insights into the history of agriculture in
southern South America. Proceedings of the Royal Society Biological Sciences 274:
545554. doi:10.1098/rspb.2006.3747.

38.

Smith BD (2001) Low-level food production. Journal of Archaeological Research 9: 1

43.

39.

Pearsall DM (2009) Investigating the Transition to Agriculture. Current Anthropology

50: 609613. doi:10.1086/605406.

40.

Aceituno J, Loaiza N (2014) Early and Middle Holocene evidence for plant use and
cultivation in the Middle Cauca River Basin, Cordillera Central (Colombia).
Quaternary Science Reviews 86: 4962. doi:10.1016/j.quascirev.2013.12.013.

18

41.

Ranere AJ, Lpez C (2007) Cultural diversity in late Pleistocene/early Holocene

populations in northwest South America and lower Central America. International
Journal of South American Archaeology 1: 2531.

Figure Legends
Figure 1. Ecuadorian preceramic sites and area of recent findings. (A) Approximate
location of known preceramic areas or sites in Ecuador. (B) Distribution of preceramic
localities within the Cubiln area and periphery. The dark polygon in Figure 1B represents the
approximate area where previous studies identified another 24 preceramic-like localities
(known as Cubiln sites).

Figure 2. East and north stratigraphic profiles for test units 1 and 2 at Cu-S2, and
profile projection of vertical location of studied artifacts and 14C samples. Only one
occupational surface was clearly detected in the contact layer between the bottom of stratum
IV and stratum III, while other lithic artifacts were documented near a possible surface at the
top of stratum II. In addition, more traces of human activity were recorded within the basal
matrix of stratum II to the north in Unit 1, though artifacts were distributed interruptedly and
with a heavily marked leaning placement that does not have any correspondence with the
stratigraphy recorded above it. The distribution of artifacts at lower levels of the excavation
units seems to be related to processes of humanly induced vertical displacement by unknown
intrusive actions and/or by natural re-deposition.

Figure 3. Selected starch grains recovered from lithic tools. (A-F) Starch granules of
maize with transverse tf radial rf y yf and asymmetric af fissures. D) Starch granule
19

of maize showing also radial striations (pressure damaging). (E-F) Heavily damaged starch
granules of maize showing typical double-border (db) characteristic of the species (A-C [tool
13-99], D-F [tool 13-98]). (G) Damaged starch granule of Dioscoreaceae showing diffused
Maltese cross (tool 13-98). (H) Damaged starch granule of Calathea spp. with lamellae (l)
(tool 13-98). (I

tarch granule of possible manioc with v flexion lines vf and hilum h

(tool 13-99). (J) Starch granule of chili pepper with longitudinal fissure (lf) (tool 14-04). (K)
Damaged starch granule of Phaseolus spp. showing smooth lamellae and an irregular
longitudinal fissure (tool 14-05).

Figure 4. Error bar plot for minimum-maximum, and mean length size of maize,
comparing archaeological starches (secure and tentative identifications combined, this
study) to 23 modern reference specimens.

Supporting Information Legends

Figure S1. Studied lithic tools. (A-A1) Expedient tool 13-98, milling stone base fragment;
siliceous rock. (B-B1) Formalized tool 13-99 bec type scraper; siliceous rock. (C-C1)
Formalized tool 14-01, scraper flake with a retouched and concave active border; siliceous
rock. (D) Formalized tool 14-02, burin flake with scraper wear signs in its longitudinal
borders, siliceous rock. (E-E1) Formalized tool 14-03, scraper with a concave active border;
siliceous rock. (F) Expedient tool 14-04, scraper flake with use-wear microflaking; siliceous
rock. (G) Expedient tool 14-05, scraper chip with use-wear microflaking, quartzite type rock.
(H) Formalized tool 14-06, scraper flake with a retouched and concave active border, and a
podlid fracture (fire damaging); siliceous rock. (I-I1) Formalized tool 14-07, terminal scraper
with convex active border; chalcedony rock. (J) Formalized tool 14-08, scraper flake with
retouched and concave active border; siliceous rock. Scale lines for each tool represent 5 cm.
20

Figure S2. Starch grains from 4 modern Ecuadorian maize landraces (Laboratorio de
Investigacin, Instituto Nacional de Patrimonio Cultural [LIINPC] reference collection).
(A) Blanco blandito redondeado white and soft (floury) endosperm. These starches are
mostly regular (spherical to oval) with some few polygonal starches. Hilum is open and
commonly visible, and fissures (usually T shape and transverse are observed in a few
cases. A prominent double-border is frequent. (B) Morochillo white, quite translucent hard
(flint) endosperm. Starches are mostly regular and the hilum is sometimes open, but is
commonly similar to the wide and dark depression registered at the hilum area on starches
submitted to the toasting of maize kernels. Fissures of Y T and transverse-line shapes are
very common. A prominent double-border is very frequent. (C) cf. Mishca yellow and soft
(floury) endosperm. Starches are mainly regular (oval), though bumpy surface is usual on
bigger oval or lightly irregular starch grains. Fissures are recurrent and transversal or
restricted lineal fissures are the most frequent ones, but there are other fissure shapes such as
T Y and radial or stellate. Different from other maize starches a prominent doubleborder is not common. (D) Tusilla yellow-orange, translucent hard (flint) endosperm.
Different from other modern Ecuadorian maize starches from mid- to high elevations, these
are the only ones in our reference collections that are mainly irregular in shape (oval, strongly
undulated, and quadrangular to hexagonal). Hilum is evident, but it is sometimes partially
closed or very smooth. Transverse, radial and restricted-transverse fissures are very common.
Like in other maize starches, a prominent double-border is evident. In sum, standard
characteristics of our modern maize starches show that shapes are usually spherical to
polygonal and display multiple pressure facets according to matrix (endosperm) hardness
[29]. The hilum is commonly open, slightly irregular, and most common in soft endosperm
maize kernels. amellae are generally absent and fissures with different patterning Y T
transversal, and sometimes radial) are common, especially in starches with dried or hard

21

(flint) kernels. One of the main diagnostic features defined for maize starch grains is the
presence of a double-border. Size is slightly variable among the different maize landraces
although a general range of 4-28m, with a mean size of 13.6 4m for 19 indigenous
landraces has been documented from our reference collections.
Figure S3. Starch grains from 4 modern bean (Phaseolus spp.) specimens (LIINPC,
reference collection). (A) Phaseolus lunatus - brown seed variety. These starches are mostly
oblong to oval, with kidney-shapes commonly present. Hilum is not observable though it is
usually centric. General size range is from 8.35 to 49.02m with a mean size of 28.51
(10.51). Lamellae consist in prominent layers of concentric undulated rings. Fissures are
mostly longitudinal lines lf in figure above straight or undulated that generally posses
thinner parallel or lightly diagonal smaller striations. No pressure facets have been registered.
(B) Phaseolus lunatus - dotted seed variety. These starches are mostly oblong to oval, with
kidney-shapes commonly present. Compound starches are commonly present. Hilum is not
observable though it is usually centric. General size range is from 9.76 to 50.24m with a
mean size of 30.01(9.24). Lamellae consist in prominent layers of concentric undulated
rings. Fissures are mostly longitudinal lines (straight or undulated) that generally posses
thinner parallel or lightly diagonal smaller striations. No pressure facets have been registered.
(C) Phaseolus vulgaris black seed variety. These starches are mostly oblong to oval, with
kidney-shapes commonly present. Compound grains are usual. Hilum is not observable
though it is usually centric. General size range is from 8.11 to 45.48m with a mean size of
24.43(8.82). Lamellae, although smoother than previous specimens, consist of the same
pattern of concentric undulated rings. Fissures are mostly longitudinal and straight lines. No
pressure facets have been registered. (D) Phaseolus vulgaris - boca negra, black variety.
These starches are mostly oval to oblong, with kidney-shapes commonly present. Compound
starches are fairly common. Hilum is not observable though it is usually centric. General size
22

range is from 12.27 to 26.93m with a mean size of 19.82 (4.77). Lamellae consist of
prominent layers of concentric undulated rings. Fissures are mostly irregular, less pronounced
than in the other specimens described above, and sometimes they consist of radial lines
combined with thinner asymmetric striations. No pressure facets have been registered.
Archaeological starches tentatively or securely identified as Fabaceae or Phaseolus spp. at
Cu-S2 coincides in shape, size, lamellae and sometimes in fissure characteristics with
previously described modern starches. However, pressure damage on archaeological starches
made it impossible to rotate them for exploring and validating identifications to a lower
taxonomic level. Table 4 show that size ranges of archaeological Fabaceae/Phaseolus starches
produced highly comparable metric data for proposing confident identifications at the three
levels here considered.
Figure S4. Starch grains from 4 modern yam (Dioscoreaceae) specimens (LIINPC, and
Pagn-Jimnez [17] reference collections). (A) D. glandulosa starch grains are mainly
single with oval and trasovate shapes, and lightly undulated margins. Proximal section is
wider than distal. Hilum is mostly closed and eccentric. General size range is from 3.6 to
64m with a mean size of 27.7 (16.6). Concentric rings are the main feature of lamellae.
Fissures are not present and Maltese cross is mainly an eccentric X shape with lightly wavy
arms. A small single pressure facet is common at the distal section. (B) Dioscorea spp. #4
produces single and compound starches with triangular shapes and obtuse angles. They are
wider in the proximal section. Hilum is closed, difficult to see, and eccentric. General size
range is from 9 to 49m with a mean size of 24 (9.5). A combination of concentric circles
and angular rings are the main lamellae characteristic. Fissures are not present and Maltese
cross is commonly an eccentric X shape with curved arms. A small single pressure facet is
common at the distal section. (C) Dioscorea spp. #5 possesses single and compound starches
with variable triangular shapes and less frequent trasovate and polymorphs grains made up by
23

two or more granules. Proximal section is wider than the distal. Hilum is closed and eccentric.
General size ranges is from 9 to 58m with a mean size of 22 (10.8). Combined concentric
circles and angular rings form the prominent lamellae on these starches. Fissures are not
present and Maltese cross is mainly an X shape with wavy arms. At the distal section one or
two small pressure facets are common. (D) Dioscorea altissima (syn. D. samydea and D.
maranonensis in Ecuador and Per) produces variable triangular shapes, notably wider at the
distal section. Hilum is closed and eccentric at the narrower proximal section. General size
range is from 15 to 75m with a mean size of 38 (13). Concentric rings with wide obtuse
angles at the observable end are the key feature of the lamellae. Curved-line fissures can be
frequent between margin and hilum at the proximal section, while the Maltese cross has an
X shape with three curved arms and one heavy wavy arm. No pressure facets were noted in
these starches. Starches from D. altissima possess many of the main characteristics registered
in one of the archaeological Dioscorea specimens identified at Cu-S2 (see Figure 3, G).
Figure S5. Starch grains from two modern calathea (Calathea spp.) specimens (LIINPC,
and Pagn-Jimnez [17] reference collections). (A) Starches from the rhizome of Calathea
spp. (wild, S. Domingo de los Tschilas) are mostly triangular with some variants. A
pronounced convex margin is common at the distal section with a narrower proximal section.
Hilum is closed and eccentric, hardly observable. General size range is from 3.5 to 28.3m
with a mean size of 13.6 (7.02). Lamellae consist of regular and very smooth concentric
rings. No fissures were noted and the Maltese cross is eccentric, mainly a cross shape with
straight arms or wavy arms in a few occasions. No pressure facets were registered. (B)
Starches from the tubers of Calathea allouia (Puerto Rico). Tubers stores quite regular
triangular starches with obtuse angles and lightly undulating margins. The proximal section is
always narrower than the distal one when they are in centric position. Hilum is eccentric and
sometimes open. General size range is from 8 to 40m with a mean size of 28 (8.6).
24

Lamellae begin with a single circle followed by regular concentric rings. Fissures will be
common in the form of small and restricted transverse lines just over the hilum. Maltese cross
with an eccentric X shape and straight arms is the most frequent, although centric cross
shape with straight arms have also been noted. No pressure facets are present. Archaeological
starch grain identified as Calathea spp. does not match the cultivar species starches of C.
allouia. However, shape, lamellae, Maltese cross and size range are closer to the modern wild
Calathea specimen described above. Improvement of this specific identification will require
the acquisition of more wild Marantaceae and Calathea specimens from lowland and midelevations areas.
Figure S6. Modern starch grains of manioc (Manihot esculenta Crantz) from Ecuador
(LIINPC reference collection). Among the diagnostic features of the starches stored in the
tuberous roots of manioc, the bell-shape with two to four pressure facets is the most
conspicuous. This starch type also possesses eccentric Y and stellate or radial fissures in
the proximal section see examples of ds or diagnostic starches above . ther common
shapes registered for these starches are the oval with 5 or 6 marginal and restricted pressure
facets, and a truncated-shape with a single pressure facet at the distal section, together with a
wide flexion line that departs from the hilum and ends at the distal section see arrows
above). General size range for all these starches is from 6.7 to 37.3m with a mean size of
17.5 (8). Hilum will be either open or closed and is usually found at centric or lightly
eccentric position. Maltese cross is mostly a centric cross shape with straight arms, although
eccentric X or cross shapes with wavy arms are present. ther plants such as sweet potato
(Ipomoea batatas), produces starch grains similar to the truncated-shape with flexion
lines described before, though differences are also contrasting among them. Sweet potato
produces very low quantities of this starch type when compared to manioc, though among
manioc they fall between 17 to 20m and lamellae is not obvious, while in sweet potato they
25

are not bigger than 14m and lamellae is prominent. Tentative identification of manioc in this
article is based on the recovery of a single starch grain almost identical to the truncated-shape
specimen described above and because it fits the mean size described for the modern species
starches (17.5m).
Figure S7. Starches from archaeological Indian potato (cf. Sagittaria spp.), and modern
Sagittaria latifolia, and S. lancifolia (LIINPC, and Pagn-Jimnez [17] reference
collections). (A-B) Archaeological starch grains from cf. Sagittaria spp. (Cu-S2, artifact 1399). (C) Modern starches of Sagittaria latifolia (Pastaza, Ecuador). (D) Modern starches of
Sagittaria lancifolia (Puerto Rico). Starches from the rhizome of Sagittaria latifolia (wild,
Pastaza, Ecuador) are mostly oval with undulated margins, although trasovate shapes with a
rounded distal end is less common. Hilum is typically closed and eccentric, or quite centric on
a few occasions. General size range is from 4.6 to 26.8m with a mean size of 14.8 (5.8).
Lamellae are smooth and consist of regular concentric circles. Fissures are very few and the
most frequent is a small transverse fissure over the hilum. Other fissures, much less common,
has an H or T shape at the proximal section. Maltese cross is mainly an eccentric cross
shape with lightly wavy arms. However it is recurrent with an eccentric X shape also with
wavy arms. Smooth, small and marginal single pressure facets were registered in just a few
cases. Archaeological starch grains tentatively identified as Sagittaria spp. does not match
diagnostic and bigger starch grains documented for S. lancifolia [29], but they possess general
features that coincide closely with S. latifolia starches previously described. Both
archaeological starches are oval to transovate; they fall within the range size of S. latifolia
(Table 4) and mean size is statistically the same. Maltese cross in archaeological starches is an
X shape with wavy arms and the proximal section is lightly narrower than the distal one
similar to many starch grains of S. latifolia. Both archaeological starches show signs of
damaging by pressure and their surfaces are partially rough, different from the unaffected
26

starches of S. latifolia here studied. So, this final observation was the main issue that hindered
the ascription of theses starches to a secure taxonomic level.

27

Tables
Table 1. Starch grains recovered from tools at Cu-S2, with sample proveniences and

Unit 1:
*8078 7959
Unit 1:
*8078 7959
Unit 1:
*8078 7959

Unit 1, St. III

10-20cmbd
Unit 1, St. III
10-20cmbd
Unit 1, St. III
10-20cmbd

Unit 2:
*8057 7927
Unit 1:
9463 9233
Unit 2:
*8057 7927
Unit 1:
9463 9233
Unit 1:
16309 15878
Unit 1:
16309 15878

Unit 1, St. II
20-30cmbd

Unit 1:
>43500
Unit 1:
>43500

Unit 1, St. II
50-60cmbd
Unit 1, St. II
60-70cmbd

Total

Unit 1, St. II
20-30cmbd

Unit 1, St. II
30-40cmbd
Unit 1, St. II
45cmbd

formal. tool, scraper

flake with retouched
borders
formal. tool, scraper
formal. tool, scraper
formal. tool, scraper
flake with retouched
borders
formal. tool, burin with
use-wear signs (scraper
type) in its longitudinal
borders
formal. tool, scraper
flake with retouched
borders
expedient tool, scraper
chip with use wear signs
expedient tool, scraper
flake with use wear
signs
expedient tool, milling
stone base fragment
formal. tool, bec type
scraper

14-03

14-07
14-08

3
4

14-06

13-98

1
1

13-99
3

15
5

c. 8

8 (+c.8)

15

10

22

10

47

1
1

14-05

Total

N.I. Tuber

N.I.

14-02

14-04

cluster cf. Zea

mays

cf. Sagittaria
latifolium

cf. Manihot
esculenta

cf. Fabaceae

Dioscoreaceae

Fabaceae

Phaseolus spp.

Calathea spp.

Capsicum spp.

Sample No.
14-01

Zea mays

Unit 1, St. III

10-20cmbd

cf. Zea mays

Unit 1:
*8078 7959

Tool type

Provenance

Associated
14
C dates
(2 cal. yrs. BP)

associated 14C dates.

25

c. 60

11

48 (+ c.60)

25

78

c. 68

37

161 (+ c. 68)

Table notes: 14C dates with an asterix (*) are used here as confident chronological markers for the upper strata
lying uniformly above all the artifacts studied. All the artifacts below this stratum are at least of equal or older
contexts. 14C dates with are chronological markers which do not fit with the associated identified taxa due to
discrepancies regarding the accepted range of dates for the initial domestication of maize (~10000-9000 BP). 14C
dates with an have been discarded. All dates in this report were calibrated using Calib Radiocarbon
Calibration Program, version 7.0.1, with Intcal13 and Southern Hemisphere 13 corrections when it was required.

28

Table 2. Chronological dataset for Cu-26, Cu-27, and Cu-S2, Cubiln sites, Ecuador.
Site

Radiocarbon
Lab. Code

Provenance

Uncal.
Date

Dated
material

1 (BP) and
relative area

2 (BP) and
relative area

Cu-27

Ki-1640

NA

10500130

charcoal

12553 12362
(0.451825)

12683 11946
(0.993306)

[10]

Cu-27

Ki-1642

NA

10330170

charcoal

12412 11771

12560 11393
(0.993192)

[10]

Reference

(1.0)
Cu-26

Ki-1859

NA

9100120

charcoal

10407 10130
(0.802247)

10521 9856
(0.969113)

[10]

Cu-26

Ki-1860

NA

9160100

charcoal

10421 10194
(0.991429)

10571 10125

[10]

(0.9482)
Cu-S2

Cu-S2

Beta-362876

Beta-364214

Test Unit 1,
Stratum II, Level
40-50cmbd

1343060

Test Unit 1,
Stratum II, Level
20-30cmbd

836040

organic
sediments

charred
remains

16219 16009

16309 15878

(1.0)

(1.0)

9421 9287

9463 9233

(1.0)

(0.925786)

This study

This study

Cu-S2

Beta-364213

Test Unit 1,
Stratum III, Level
10-20cmbd

726040

charred
remains

8051 - 7967
(0.862641)

8078 7959
(0.714983)

This study

Cu-S2

Beta-362878

Test Unit 2,
Stratum III, Level
20-30cmbd

721040

charred
remains

8014 7952

8057 7927
(0.938595)

This study

Test Unit 1,
Stratum II

>43500

NA

This study

Cu-S2

Beta-362877

(1.0)
charred
remains

NA

29

Table 3. Modern and archaeological starch grain size ranges of maize specimens. Some
modern maize landraces, and kernel variability, were submitted to different grinding
processing.

Zea mays
Modern, control samples from the Circum-Caribbean [17]
i
Mature, dry and hard kernels soaked for 24 hours before grinding
a
a. Pollo (CIMMyT Id#:3106)
b. Early Caribbean (CIMMyT Id#:1347)
c. Negrito de Colombia (CIMMyT Id#:3199)
d. Cateto cristalino (CIMMyT Id#:4113)
e. Chandelle (CIMMyT Id#:3879)
f. Tuon (CIMMyT Id#:5495)
Modern, control samples from Ecuador (this study) ii
Dry and hard kernels, unsoaked
g. Tuxpeo/Arizona (LIINPC b)
h. Canguil puntiagudo (LIINPC)
i. Blanco blandito puntiagudo (LIINPC)
j. Blanco blandito redondeado (LIINPC)
k. cf. Tusilla (LIINPC)
l. cf. Mischca (LIINPC)
m. Chulpi Ecu (LIINPC)
n. Morochillo (LIINPC)
o. Morocho (LIINPC)
p. Racimo de uva (LIINPC)
q. Sangay (LIINPC)
r. Triunfo (LIINPC)
s. Trueno (LIINPC)
Modern, control samples after experimentation iii
Green to dry kernels soaked, or not (marked with * below), for 1
hour before grinding [29]
t. Nal-Tel,mature, dry and hard (CIMMyT Id#: 815)
u. Pollo, mature, dry and hard (CIMMyT Id#: 3105)
v. Pollo, semi-mature and partially hard (CIMMyT Id#: 3105)
w. Pollo*, green and soft (produced after CIMMyT Id#: 3105)
Archaeological starch grains from Cu-S2, all artifacts (this
study) iv
Zea mays
cf. Zea mays

Range of sizes in m

Mean sizes in m

No. measurements
taken

2-28
3-20
5-20
3-18
2-20
1-18

13 3.9
13 3.6
12.3 3.3
10.3 3.1
12.3 3.2
12 3.2

116
101
107
107
89
109

4-23
3-26
5-20
10-26
6-26
3-26
9-17
6-26
4-24
4-20
3-18
5-18
6-20

12.79 4.62
15.47 5.03
12.96 3.68
17.1 4.68
15.8 5.14
18.2 5.93
12.9 1.99
16.7 5.2
13.3 4.71
12.3 4.05
13.3 3.5
12.5 2.7
13 3.3

20
20
20
20
20
20
20
20
20
20
20
20
20

11-41
10-38
7-34
5-25

21.8 7.7
23.2 6.6
20.8 5.7
12.1 4.7

60
60
60
60

10-24.87
12.5-26.25

16.27 2.89
18.92 3.21

78
25

Table notes:
i. After soaking, kernels of control samples from the Circum-Caribbean [17] were gently ground for 15 seconds
with a marble mortar and pestle to avoid overly damaging the starch grains.
ii. Unsoaked kernels of control samples from Ecuador (this study) were opened and gently scraped with a
scalpel.
iii. After soaking, kernels of experiment samples [29] were ground intensively for five minutes with a marble
mortar and pestle in order to examine patterns of damage due to pressure.
iv. Maize starch clusters are excluded.
a. CIMMyT = Centro Internacional de Mejoramiento de Maz y Trigo, Mexico.
b. LIINPC = Laboratorio de Investigacin, Instituto Nacional de Patrimonio Cultural, Ecuador.

30

Table 4. Size ranges of starch grains identified in this study compared to modern specimens*
Taxa (Identified archaeological
starches)

Size range in m
(min-max. length) of
archaeological
starches

Mean size in m of
archaeological
starches

Total no. of
identified
archaeological
starches

Taxa (Modern specimen starches)

cf. Manihot esculenta

17.50

17.50

Manihot esculenta

Zea mays

10 24.87

16.27 2.89

78

See Extended Data Table 3

cf. Zea mays

12.5 26.25

18.92 3.21

25

Capsicum spp.

16.34 29.96

24.38 7.14

Phaseolus spp.

27.70 31.88

29.79 2.96

Size range in
m (min-max
length) of
modern
specimens

Mean size in m of
modern specimens

Total no.
measurements
taken in modern
specimens

References (modern
specimens)

Domesticates
6.7 37.3

17.16 8

20

This study

18.7 42
13.5 41.7
2 6.1
8.1 45.5
12.3 26.9
8.4 49
9.8 50.2

29.3
25.1
3.5
24.4 8.8
19.8 4.8
28.5 10.5
30 9.2

25
25
50
20
20
20
20

[24]
[24]
[24]
This study
This study
This study

Cultivars or transitional to full

domesticates
Capsicum annum annum
C. baccatum baccatum
C. annum aviculare
Phaseolus vulgaris
P. vulgaris, Boca Negra black

Fabaceae

20.77 32.29

27.42 5.96

P. lunatus, brown
P. lunatus, mottled
Above (Phaseolus spp.)

cf. Fabaceae

20.99 26.47

23.73 3.87

Above (Phaseolus spp.)

Calathea spp.

22.50

22.50

Calathea spp. S.Domingo Ts

C. allouia

Dioscoreaceae

38.75 49.5

44.13 7.6

D. glandulosa
D. spp. #4
D. spp. #5
D. altissima

cf. Sagittaria latifolia

13.75 18

15.88 3.0

Sagittaria latifolia
Sagittaria lancifolia

This study

Wild
3.5 28.3
8 40

13.6 7.02
28 8.6

20
126

This study
[17]

3.6 64
9 49
9 58
15 75

27.7 16.6
24 9.5
22 10.8
38 13

20
20
20
126

This study
This study
This study

4.6 26.8
11 79

14.8 5.8
54 8

20
30

[17]
This study
[29]

Table note: * Archaeological starch clusters are excluded.

31

Table 5. General morphology and other surface features (%) of recovered maize starch
grains.
Tool
No.

Shape

Regular
(spherical to
regular oval)

Hilum

Fissure
variants *

14-01

12

Irregular
(irregular
oval to
polygonal)
88

50

25

14-03

33

67

56

22

14-07

100

75

14-08

100

100

14-02

100

100

14-06

100

100

14-05

100

33

67

Tra

eX

Cr

Rad

13

13
22

Potential damaging
vector

Circ

raL

11

25
50

dCr

Press.

13

50

50

44

56

25

100

50

50

Heat

25

25
50

50

100

14-04

25

75

38

38

13-98

34

66

19

33

13-99

44

56

28

22

33
1
3
3

3
3

13

25

44

13

50

16

Total no. of
maize
starches per
sample **

Nothing

100

33

67

38

62

22

32

22

32

78
75

Table note: *More than one fissure variant can be present in the same starch grain if rotated. **Secure and tentative
identifications have been combined. Fissure variants legend: Tra=transverse, m=m shape eX=expanded X
shape, Cr=cross, Rad=radial, Circ=circle, raL=ramified line, dCr=deep cross.

32

Figure 1
Click here to download high resolution image

Figure 2
Click here to download high resolution image

Figure 3
Click here to download high resolution image

Figure 4
Click here to download high resolution image

Supporting Information Figure 1

Click here to download Supporting Information: JRPJ-FigureS1.tif

Supporting Information Figure 2

Click here to download Supporting Information: JRPJ-FigureS2.tif

Supporting Information Figure 3

Click here to download Supporting Information: JRPJ-FigureS3.tif

Supporting Information Figure 4

Click here to download Supporting Information: JRPJ-FigureS4.tif

Supporting Information Figure 5

Click here to download Supporting Information: JRPJ-FigureS5.tif

Supporting Information Figure 6

Click here to download Supporting Information: JRPJ-FigureS6.tif

Supporting Information Figure 7

Click here to download Supporting Information: JRPJ-FigureS7.tif

Supporting information
Click here to download Supporting Information: JRPJ-Original Figure 1A.jpg